During the past four years of this Program Project, studies have provided significant new insights into the cell biology of water channel (aquaporin) trafficking, the regulation of the actin cytoskeleton and associated proteins and their relationship to ion channel function, the role of GTP-binding proteins in Golgi vesicle trafficking, the interaction between extracellular matrix proteins and cell polarity, and the role of cytosolic PLA2 in cell signaling processes. Taking advantage of novel tools and model systems that have been developed during the past funding period. The first will use stably transfected LLC-PK1 epithelia cells to examine intracellular pathways, protein sorting signals and mechanisms of aquaporin trafficking, comparing the vasopressin-regulated membrane insertion of AQP2 with the constitutive pathway followed by AQP1. The second project will define the role of the actin cytoskeleton and associated regulatory proteins in vesicle trafficking, using a unique melanoma cell line that is deficient in the ABP280 actin binding protein, in conjunction with studies on the regulation of AQP2-containing vesicles in transfected LLC-PK1 cells. The third project takes advantage of the recent discovery of a class of G-protein regulating proteins called RGSs, that modulate the function of heterotrimeric G-proteins. The interactions between Gi3 and a member of the RGS family called GAIP, and MAP kinases and their role in controlling vesicle and protein trafficking will be explored using newly-developed transfected cell culture systems. The fourth project will examine H+ ATPase trafficking and recycling processes in specialized epithelial cells in the epithelium of the epididymis and vas deferens. This represents a novel and virtually unique mammalian model system in which the epithelial surface is readily accessible for real time functional measurements using ion-selective vibrating probe technology, combined with cell biological and tracer studies on the regulation of intracellular pathways of exo- and endocytosis. Finally, the fifth project will continue to elucidate the role of PLA2 and one accessory protein (PLIP) in cell signaling events, with particular emphasis on cytosol to nuclear trafficking processes. As in the previous 9 years, this Program Project brings together a group of scientists with considerable expertise in many aspects of cell biology. Molecular biology, biophysics, electrophysiology, and biochemistry which will provide for a great deal of interaction among the participants. Further interaction is provided via the centralized Microscopy Core, which plays a major role in coordinating cell biological and morphological studies for all projects. It is once again envisaged that the distinct but related series of studies proposed, together with the established and ongoing interactions of the investigators, will allow us to examine in greater depth the relatinships between the plasma membrane and the cytoplasm of epithelia cells that are important both for normal function and disease.